Camera imaging system for a fluid sample assay and method of using same

ABSTRACT

Apparatuses and for causing a point-of-care polymerase chain reaction and analyzing the polymerase chain reaction at the point-of-care, particularly when unwanted bubbles are present during the polymerase chain reaction, are described herein. In a general embodiment, a device for analysing a polymerase chain reaction in a fluid sample includes a current source configured to cause the polymerase chain reaction by heating the fluid sample within a target zone, a camera imaging device configured to record a plurality of images of the fluid sample in the target zone while the current source causes the polymerase chain reaction, and a controller configured to (i) distinguish wanted objects in the plurality of images from an unwanted object in the plurality of images, and (ii) determine whether the fluid sample tests positive or negative for a bacteria or virus based on the wanted objects.

PRIORITY CLAIM AND CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/182,992, entitled “Point-Of-Care PCR Assay for InfectiousAgents”, filed Jun. 22, 2015, and U.S. Provisional Patent ApplicationNo. 62/187,471, entitled “Point-Of-Care PCR Assay for InfectiousAgents”, filed Jul. 1, 2015, the entire contents of each of which arehereby incorporated by reference and relied upon. This application isalso related to U.S. application Ser. No. ______, entitled “Device forAnalyzing a Fluid Sample and Use of Test Card with Same”, filedconcurrently herewith under Attorney Docket No. 1958928-00006, and U.S.application Ser. No. ______, entitled “Test Card for Assay and Method ofManufacturing Same”, filed concurrently herewith under Attorney DocketNo. 1958928-00007, the entire contents of each of which are herebyincorporated by reference and relied upon.

FIELD OF THE DISCLOSURE

The present disclosure relates generally to apparatuses and methods forperforming an assay, and more specifically to apparatuses and methodsfor causing a point-of-care polymerase chain reaction and analyzing thepolymerase chain reaction at the point-of-care.

BACKGROUND OF THE DISCLOSURE

Point-of-care (POC) in vitro diagnostics tests (IVDT) have traditionallyhad two major categories, nucleic acid amplification tests (NAAT) orimmunoassay-based tests. The former directly detects the pathogen's DNAor RNA, while the latter detects antibodies or antigens generated by theimmune system response to the pathogen.

Current POC diagnostic immunoassays lack the high sensitivity andspecificity of nucleic acid amplification methods. This becomes morepronounced during the initial stages of infection, often within 168hours. Taking the case of Dengue virus in whole blood, immunoglobulin M(IgM) and immunoglobulin G (IgG) remain undetectable in the majority ofpatients until 5 and 10 days post-infection, respectively, whereasnucleic acid can be found as early as 0 to 7 days. Moreover, manyimmunoassay tests are unable to detect infectious agents until 3 monthsafter the initial onset of the infection. This delay is due to the timeit takes for the body's immune system to respond to an infection.

POC diagnostic assays developed utilizing NAATs have very highsensitivities and specificities, matching those of currently acceptedlaboratory tests. The primary mechanism of NAAT based systems is todirectly detect an infectious agent's nucleic acid, lending to thetest's ability to detect diseases within the first few days of the onsetof infection. In addition, by careful primer design, NAATs also have theability to have very high specificity and sensitivity compared toimmunoassay based testing. The largest drawback of NAATs compared toimmunoassay-based tests is the complicated equipment and/or processesrequired to prepare a sample for testing.

SUMMARY OF THE DISCLOSURE

Described herein are methods and apparatus for causing a point-of-carepolymerase chain reaction and analyzing the polymerase chain reaction atthe point-of-care, particularly when unwanted bubbles are present duringthe polymerase chain reaction. In a general embodiment, a device foranalysing a polymerase chain reaction in a fluid sample includes acurrent source configured to cause the polymerase chain reaction byheating the fluid sample within a target zone, a camera imaging deviceconfigured to record a plurality of images of the fluid sample in thetarget zone while the current source causes the polymerase chainreaction, and a controller configured to (i) distinguish wanted objectsin the plurality of images from an unwanted object in the plurality ofimages, and (ii) determine whether the fluid sample tests positive ornegative for a bacteria or virus based on the wanted objects.

In an example embodiment, the wanted objects are nucleic acid moleculesand the unwanted object is an air bubble.

In an example embodiment, the controller is configured to analyze theplurality of images by dividing the target zone into a plurality ofbins.

In an example embodiment, the plurality of bins are arranged in a gridwith a plurality of rows and columns.

In an example embodiment, the controller is configured to determinewhether the fluid sample has tested positive or negative for thebacteria or virus by selecting at least two of the plurality of binsthat overlap a cluster of wanted objects and calculating a meanfluorescence value of the at least two of the plurality of bins.

In an example embodiment, the controller is configured to exclude atleast one of the plurality of bins from the mean fluorescence valuecalculation if the at least one of the plurality of bins overlaps theunwanted object.

In an example embodiment, the controller is configured to assign aweight to at least one of the plurality of bins used in the meanfluorescence value calculation based on the proximity of the at leastone of the plurality of bins to the unwanted object.

In an example embodiment, the controller is configured to exclude atleast one of the plurality of bins if the at least one of the pluralityof bins does not meet a minimum threshold value for brightness.

In an example embodiment, the controller is configured to report aninconclusive test if the controller identifies an unwanted image in theplurality of images.

In an example embodiment, the device includes a user interface, thecontroller includes a plurality of preprogrammed analyses that can beperformed on the fluid sample, and the user interface is configured toallow a user to select at least one analysis from the plurality ofpreprogrammed analyses.

In an example embodiment, the plurality of images includes at least oneof: (i) a plurality of still images of the polymerase chain reactionrecorded by the camera imaging device over a period of time; or (ii) avideo image of the polymerase chain reaction recorded by the cameraimaging device over the period of time.

In an example embodiment, the controller is configured to distinguishthe wanted objects in the plurality of images from the unwanted objectin the plurality of images by comparing a size or shape of objects inthe plurality of images to an average size or shape of blood cells.

In a general embodiment, a device for analysing a polymerase chainreaction in a fluid sample includes a current source configured to causethe polymerase chain reaction by heating the fluid sample within atarget zone, a camera imaging device configured to record a plurality ofimages of the fluid sample in the target zone while the current sourcecauses the polymerase chain reaction, and a controller configured toanalyze the plurality of images by (i) dividing the target zone shown inthe plurality of images into a plurality of bins, (ii) selecting atleast two of the plurality of bins, and (iii) calculating a fluorescencevalue of the selected at least two of the plurality of bins.

In an example embodiment, the controller is configured to select the atleast two of the plurality of bins based on a threshold value forbrightness.

In an example embodiment, the controller is configured to exclude atleast one of the plurality of bins from the fluorescence valuecalculation if the at least one of the plurality of bins overlaps anunwanted object.

In an example embodiment, the controller is configured to assign weightsto the selected at least two of the plurality of bins for thecalculation based on the proximity of the selected at least two of theplurality of bins to an unwanted object.

In a general embodiment, a method of analysing a polymerase chainreaction in a fluid sample includes heating the fluid sample in a targetzone to cause the polymerase chain reaction, recording a plurality ofimages of the fluid sample in the target zone during the polymerasechain reaction, dividing the target zone into a plurality of bins,calculating a fluorescence value of at least two of the plurality ofbins, and determining whether the fluid sample tests positive ornegative for a bacteria or virus based on the calculated fluorescencevalue.

In an example embodiment, calculating the fluorescence value includesweighting the at least two of the plurality of bins based on proximityto an unwanted object.

In an example embodiment, calculating the fluorescence value includesexcluding at least one of the plurality of bins from the fluorescencevalue calculation if the at least one of the plurality of bins overlapsan unwanted object.

In an example embodiment, calculating the fluorescence value includesexcluding at least one of the plurality of bins based on a thresholdvalue for brightness.

In a general example embodiment, a device for analysing a fluid sampleincludes a camera imaging device configured to record a plurality ofimages of the fluid sample while located within a target zone of a fluidmicrochannel, and a controller configured to (i) distinguish wantedobjects in the plurality of images from an unwanted object in theplurality of images, and (ii) determine whether the fluid sample testspositive or negative for a bacteria or virus based on the wantedobjects.

In an example embodiment, the controller is configured to determinewhether the fluid sample tests positive or negative for the bacteria orvirus by dividing the target zone shown in the plurality of images intoa plurality of bins, selecting at least two of the plurality of bins,and calculating a fluorescence value of the selected at least two of theplurality of bins.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present disclosure will now be explained in furtherdetail by way of example only with reference to the accompanyingfigures, in which:

FIG. 1 is a top perspective view of an example embodiment of an assaydevice according to the present disclosure;

FIG. 2 is a top perspective view of an example embodiment of a test cardaccording to the present disclosure;

FIG. 3 is a cross-sectional view of the test card of FIG. 2;

FIG. 4 is a top perspective view of the assay device of FIG. 1 with thetest card of FIG. 2 inserted therein;

FIG. 5 is a cross-sectional view of the assay device and test card shownin FIG. 4;

FIGS. 6A to 6C illustrate an example embodiment of amplified imagesshowing a PCR;

FIGS. 7A to 7E illustrate an example embodiment showing how a controllercan detect bubbles and analyze a reaction even when bubbles aredetected;

FIG. 8 illustrates an example embodiment of a control method that can beused to perform and analyze a reaction according to the presentdisclosure;

FIG. 9 illustrates an example embodiment of a controller that canperform the method of FIG. 8.

DETAILED DESCRIPTION

Before describing in detail the illustrative system and method of thepresent disclosure, it should be understood and appreciated herein thatthe present disclosure relates to a rapid, high sensitivity and highspecificity, low complexity, diagnostic system 1 using nucleic acidamplification and capable of operating in low resource settings withminimal user training. The system described herein is configured, forexample, to cause and analyze polymerase chain reactions (PCR),particularly in the early stages of infection, using a low-costmicrofluidic platform employing PCR with a modified DNA polymerase.

FIG. 1 illustrates an example embodiment of a point-of-care diagnosticsystem 1 according to the present disclosure. As illustrated, diagnosticsystem 1 includes an assay device 10 with a housing 12 having a slot 14to receive a test card 100 (FIG. 2), which is an inexpensive, disposabletest card that can be used with device 10 and then discarded. Asexplained in more detail below, a fluid sample can be injected into testcard 100, and then test card 100 can be inserted into slot 14 so thatdevice 10 can power test card 100 to run an assay within test card 100without further action by the user. The resulting analysis can then bedisplayed to the user by user interface 60. Test card 100 can then bediscarded and a new test card 100 can be inserted into slot 14 and usedthe same way to run a new assay. In an embodiment, the test card isconfigured to receive about 10 μL of whole blood, the equivalent to adrop of blood obtained from a finger stick. In another embodiment, thefluid sample can be serum, urine, saliva, tears and/or the like.

Device 10 is described in more detail in U.S. application Ser. No.______, entitled “Device for Analyzing a Fluid Sample and Use of TestCard with Same”, filed concurrently herewith under Attorney Docket No.1958928-00006, the entire disclosure of which, and specifically thedevice and test card structure disclosure, is incorporated herein byreference and relied upon. Those of ordinary skill in the art willrecognize other configurations of device 10 that can be used accordingto the present disclosure.

Test card 100 is described in more detail in U.S. application Ser. No.______, entitled “Test Card for Assay and Method of Manufacturing Same”,filed concurrently herewith under Attorney Docket No. 1958928-00007, theentire disclosure of which, and specifically the test card structuredisclosure, is incorporated herein by reference and relied upon. Thoseof ordinary skill in the art will recognize other configurations of testcard 100 that can be used according to the present disclosure.

As illustrated in FIGS. 2 and 3, test card 100 includes an inlet port124, a mixing chamber 126, a capture port 128, an outlet port 130, and afluid microchannel 134. A liquid sample can be injected into inlet port124 and mixed with one or more reagent in mixing chamber 126, and thentest card 100 can be placed into slot 14 of assay device 10. Once testcard 100 has been placed within device 10, the fluid sample can bepulled though fluid microchannel 134, so that the fluid sample can beanalyzed through an analysis port 132 of test card 100. Test card 100also includes electrical contacts 122 on a bottom surface 102 thereof,which enables electrodes adjacent to fluid microchannel 134 to becontrolled to power test card 100, for example, to heat fluid withinfluid microchannel 134, track fluid flow through fluid microchannel 134and/or measure the concentration of a chemical species in the fluidsample.

FIG. 4 illustrates a perspective view of device 10 after test card 100has been placed into slot 14, and FIG. 5 illustrates a cross-sectionalview thereof. As illustrated, placement of test card 100 into slot 14aligns several of the elements of device 10 with several of the elementsof test card 100. For example, placement of test card 100 into slot 14aligns camera imaging device 20 and light source 22 of device 10 withanalysis port 132 on an upper surface 104 of test card 100, pneumatictube 40 of fluid actuation source 24 with outlet port 130 on the uppersurface 104 of test card 100, and electrical contacts 42 of electricalcontact device 26 with electrical contacts 122 on the bottom surface 102of test card 100.

As explained in more detail in U.S. application Ser. No. ______,Attorney Docket No. 1958928-00006, once pneumatic tube 40 is sealedagainst outlet port 130, a negative pneumatic force can be applied tooutlet port 130 from fluid actuation source 24. When the negativepneumatic force is applied, the fluid sample injected into inlet port124 is pulled through fluid microchannel 134 towards outlet port 130.The fluid sample however is not pulled into pneumatic tube 40 due to thepresence of capture port 128 between inlet port 124 and outlet port 130.Capture port 128 allows fluid to build up before it can reach outletport 130 and/or pneumatic tube 40, which keeps device 10 sterile andprotects the integrity of diagnostic system 1.

Test card 100 is dimensioned so that electrical contacts 122 of testcard 100 are placed into electrical contact with electrical contacts 42of electrical contact device 26 when test card 100 is fully insertedinto slot 14. With the electrical contacts 42 and 122 aligned,controller 28 can perform several functions. Before beginning an assay,controller 28 can ensure that the fluid sample has been properly pulledthrough microchannel 134 and into a target zone of microchannel 134 bymeasuring the capacitance of the fluid sample upstream and/or downstreamof the target zone. Once it is determined that fluid is located withinthe target zone of microchannel 134, controller 28 can control powersource 30 to apply a current to electrical contacts aligned with thetarget zone to heat the fluid sample located within target zone andcause a reaction such as a PCR to occur.

The alignment of camera imaging device 20 over analysis port 132 on theupper surface 104 of test card 100 allows controller 28 to analyze oneor more reaction within fluid microchannel 134 while controlling thereaction. Camera imaging device 20 is configured to record a pluralityof images of the fluid sample within the target zone so that the imagescan be analyzed in real time. The plurality of images can include, forexample, a plurality of still images and/or a video image. Cameraimaging device 20 is therefore configured to record the reaction of thefluid sample by taking a series of still images of the fluid samplewithin the target zone during a reaction or by taking a video of thefluid sample within the target zone during a reaction. As explained inmore detail below, the plurality of images can be used, for example, toanalyze the physical and/or chemical characteristics of cells within thefluid sample.

The target zone can be anywhere along fluid microchannel 134 or can bebranched off of fluid microchannel 134. Example embodiments of targetzones are described in more detail in U.S. application Ser. No. ______,entitled “Test Card for Assay and Method of Manufacturing Same”, filedconcurrently herewith under Attorney Docket No. 1958928-00007. In anembodiment, the target zone can be located at a central portion of fluidmicrochannel 134 and fluid microchannel 134 can include, for example, acapacitance sensor upstream and/or downstream of the target zone todetermine whether fluid has been pulled through fluid microchannel 134and into the target zone. In an embodiment, microchannel 134 can includea plurality of target zones to perform a plurality of assays at the sameor different times, for example, to improve the reliability of theassays. In an embodiment, controller 28 can be programmed to analyze aspecific section of microchannel 134 as the target zone, or controller28 can determine the target zone based on the plurality of images takenby camera imaging device 20.

FIGS. 6A to 6C illustrate an example embodiment of amplified imagesshowing a PCR. FIG. 6A illustrates a fluid sample that has beeninitially loaded into target zone 166, with a few nucleic acid molecules50 separated by large distances. FIG. 6B shows the fluid sample duringthe PCR, when clear colonies 52 of nucleic acid molecules 50 can beviewed because the diffusion speed of the molecules is slower than thespeed required for the molecules to travel the distance betweencolonies. FIG. 6C shows the end of the PCR, when the colonies 52 are nolonger clearly visible.

From the number of colonies shown in FIG. 6B, controller 28 candetermine whether the fluid sample tests positive or negative for aspecific virus or bacteria based on a known titer value for the specificvirus or bacteria. The titer value corresponds to the highest dilutionfactor for the specific virus or bacteria that yields a positive testresult. By counting the colonies in FIG. 6B, and by comparing the countwith the known titer value, controller 28 can determine a positive ornegative test result for the fluid sample. The known titer value can becompared through a variety of methods. In an embodiment, a statisticalanalysis can be utilized to say with certain probability that the resultwill fall under a certain titer value. This can be achieved by running astatistically significant amount of controlled samples under controlleddilutions and generate a standard curve based on the crossover thresholdvalue of each (point at which the curve starts to increase).

In an embodiment, camera imaging device 20 includes a high sensitivityand dynamic range complementary metal-oxide semiconductor (CMOS) camerasensor which allows for general imaging of a reaction within target zone166 of fluid microchannel 134 of test card 100. In an embodiment, theCMOS camera sensor enables camera imaging device 20 to image the entireanalysis port 132 of test card 100. Although in the illustratedembodiment only a single target zone 166 within a single microchannel134 is shown, camera imaging device 20 is configured to image aplurality of target zones and/or a plurality of microchannels should theuser insert such a test card 100 into device 10. Examples of alternativeembodiments of test cards 100 are disclosed in U.S. application Ser. No.______, entitled “Test Card for Assay and Method of Manufacturing Same”,filed concurrently herewith under Attorney Docket No. 1958928-00007.

In the illustrated embodiment, light source 22 is configured to projecta fluorescent excitation light on target zone 166 of fluid microchannel134 during a PCR, while camera imaging device 20 is recording stilland/or video images of the PCR. In an embodiment, the fluid sample hasbeen mixed with a fluorescence reagent in mixing port 126, before thefluid sample is pulled into target zone 166. By illuminating target zone166 with a fluorescence excitation light, controller 28 can recordfluorescence measurements from the images taken by camera imaging device20. The fluorescence images can be analyzed to determine the incrementalincreases in fluorescence during each PCR cycle. This allows thedetermination of the PCR amplification curve. Additionally, the imagescan be used during melt analysis to determine the incremental decreasein fluorescence as temperature is increased. Incremental change influorescence can be determined by taking the difference in pixelintensity between successive images.

For general fluorescence measurements, for example, a statisticalformulation of the PCR can be achieved by subdividing the target zone166 and measuring florescence at various locations in the target zone166. This yields highly-accurate and consistent crossover thresholdvalues for the real-time PCR based on titer values. Once the PCR iscomplete, a melting curve analysis allows for error checking, ensuringthat the correct amplicon has been amplified during the PCR, to reducethe likelihood of a false-positive test.

Camera imaging device 20 also enables controller 28 to record a varietyof other measurements in addition to fluorescence measurements. Forexample, controller 28 can determine turbidity and object detectionusing the images taken by camera imaging device 20. The turbidity can bedetermined by using the device LEDs to apply incident light to a testcard microchannel and/or target zone. The amount of scattered light canbe measured by the camera allowing a numerical value of relativeturbidity to be measured. The turbidity can be used to determine theoutcome of various different reactions, such as measuring thereactiveness of proteins during an ELISA test (colorimetry measurement)or measurement of water quality. The object detection can be used, forexample, to determine whether air bubbles formed in target zone 166during the PCR and potentially destroyed the integrity of the PCRanalysis. By imaging the entire analysis port 132 of test card 100,camera imaging device 20 is able to greatly improve accuracy by allowingfor various advanced image-processing algorithms to be applied.

With respect to object detection, camera imaging device 20 can be usedto detect both wanted and unwanted objects within microchannel 134. Inan embodiment, the wanted objects are cells such as red and white bloodcells in the fluid sample to be analyzed during the reaction. Controller28 can count the wanted objects by matching the cell size and cell shapeof objects in the images from camera imaging device 20 to the averagesize and shape of specific blood cells, allowing controller 28 to count,for example, the number of red and white blood cells in the fluid sampleat target zone 166. Controller 28 can also distinguish the red and whiteblood cells from other unwanted objects that do not match the averagesize and shape of specific blood cells.

If controller 28 detects an unwanted object, controller 28 can determinethat the PCR has failed or is indeterminate, and can instruct a userthat an additional PCR should be run on the same or a different testcard. If the PCR is run on the same test card, target zone 166 should becleared of fluid, and then new fluid should be pulled from mixingchamber 126 into target zone 166.

In an embodiment, the unwanted image is an air bubble. In an embodiment,controller 28 can detect air bubbles because air bubbles will expand inthe target zone 166 while the PCR reaction occurs. Red and white bloodcells, on the other hand, multiply but do not increase in size.Controller 28 can therefore determine the presence of an air bubble intarget zone 166 by noting a change in size of an object across aplurality of subsequent images from the plurality of images. In anotherembodiment, controller 28 can determine the presence of an air bubble bynoting that an unwanted object does not match the average size and shapeof blood cells. In another embodiment, controller 28 can determine thepresence of an air bubble due to there being no fluorescence inside ofthe bubble by looking for a change in the gradient of fluorescence.

In some cases, a bubble formed within microchannel 134 will remain smalland will not affect any fluid flow or reaction occurring withinmicrochannel 134. When heat is applied to target zone 166, however, theheat can cause the bubble to expand due to the large thermal expansioncoefficient of the bubble.

FIGS. 7A to 7E illustrate an example embodiment showing how controller28 can detect unwanted objects such as bubbles and analyze a reactioneven when bubbles are present. In some cases, unwanted objects may causethe reaction to fail completely and need to be rerun. A failure mightoccur if there are too many bubbles to compensate for. In FIGS. 7A to7E, however, controller 28 can proceed with the reaction and analysiseven with an air bubble present.

FIG. 7A illustrates an example embodiment of target zone 166 before anyfluid has entered the target zone. As illustrated, controller 28 hasdivided target zone 166 into a plurality of bins 200. In the illustratedembodiment, controller 28 has divided target zone 166 into six rows(labeled A to F for ease of reference) and twelve columns (labeled 1 to12 for ease of reference) to create seventy-two bins 200, but those ofordinary skill in the art will recognize that any number of bins 200 canbe created within a target zone 166. It should be understood that thebins 200 are created virtually by controller 28, and there are nophysical barriers separating the bins 200 within target zone 166.

FIG. 7B illustrates target zone 166 after receiving a fluid sample butbefore a reaction has occurred. As illustrated, there are a minimalnumber of nucleic acid molecules 202 in the fluid sample before thereaction. In the illustrated embodiment, bins B7, C3, C10, D8 and E2contain nucleic acid molecules 202.

FIG. 7C illustrates target zone 166 during a PCR. As illustrated, themolecules shown in bins B7, C3, C10, D8 and E2 in FIG. 7B havemultiplied by diffusion. Clear colonies 204 of nucleic acid molecules202 can be viewed because the diffusion speed of the molecules is slowerthan the speed required for the molecules to travel the distance betweencolonies. Bins A6, A7, B3, B6, B7, C3, C4, C9, C10, D2, D3, D7, D8, E1,E2, E3, E8 and E9 each contain nucleic acid molecules 202 in FIG. 7C.

FIG. 7D illustrates bins 200 that have been selected by controller 28 tobe used in calculations for amplification and melt curves and for thecorresponding analysis. In the illustrated embodiment, the colonies 204of nucleic acid molecules 202 are visualized by controller 28 based onlocalized increases in brightness in each bin. If a bin 200 reaches athreshold value for brightness, then that bin's fluorescence value isused in calculations for amplification and melt curves and for thecorresponding analysis. If a bin 200 does not reach the threshold valuefor brightness, then that bin 200 is excluded from the calculations foramplification and melt curves and for the corresponding analysis. Inorder to get a final value for fluorescence of the entire target zone166, the arithmetic mean is taken between all of the selected bins 200.In the illustrated embodiment, bins A6, A7, B6, B7, C3, C9, C10, D2, D3,D7, D8 and E2 have been selected for use in calculations foramplification and melt curves and for the corresponding analysis, whilebins B3, C4, E1, E3, E8 and E9 have been excluded even though theycontain nucleic acid molecules because they do not meet the thresholdfor brightness.

FIG. 7E illustrates how an unwanted object such as a bubble affects theanalysis by controller 28. In the illustrated embodiment, a bubble 206has been detected by controller 28 in bins C3, C4, C5, C6, D3, D4, D5,D6, E3, E4, E5, E6, F3, F4 and F5. Even though bins C3 and D3 met thethreshold value for brightness, controller 28 has determined that binsC3 and D3 should be excluded from the amplification and melt curves andcorresponding analysis due to the presence of bubble 206. Controller 28has also determined that bins D2 and E2 should be given a lower weightduring calculations due to their proximity to bubble 206.

Based on the above analysis, controller 28 can average the fluorescencevalues of the selected bins 200. If controller 28 does not detect anybubbles in the images taken by camera imaging device 20, then controller28 calculates the fluorescence using the following equation:

${F = \frac{\sum_{i = 1}^{n}x_{i}}{n}},$

wherein n is the number of bins 200 used in the calculation, and x_(i)is the individual bins in the grid utilized by the algorithm.

If one or more bubble is detected in target zone 166, then controller 28calculates the fluorescence using the following equation:

${F = \frac{\sum_{i = 1}^{n}{a_{i}x_{i}}}{n}},$

where a_(i) is the bin's weight, which is determined by the bin'sproximity to the bubble. In an embodiment, a_(i) should be greater thanzero and less than or equal to one (0<a_(i)≦1).

In an embodiment, the a_(i)'s may all have an equal weight of 1 whenthere are no bubbles present. In an embodiment, the a_(i)'s value candecrease proportionally as the bubble gets closer to the bin.

In an embodiment, the amplification curve and melt curve are created bymeasuring the absolute value of the fluorescence of each pixel insuccessive images taken either during each cycle (for the amplificationcurve) or as temperature is slowly increased over time (for the meltcurve). In the case of an amplification curve, which shows theincremental change in fluorescence during the amplification stage of aPCR, a PCR crossover threshold value can be determined, and controller28 can look for when the fluorescence value has increased past a certainthreshold value and when the first derivative of fluorescence withrespect to cycle number is at a maximum. In the case of a melt curve,the incremental change in fluorescence can give controller 28 themelting temperature of the amplified DNA. Mathematically, controller 28looks for when the first derivative of the fluorescence with respect totemperature is at a minimum, and the second derivative of thefluorescence with respect to temperature is equal to zero.

The above detection and analysis is useful for a fluorescence based PCRor cytometry analysis, but those of ordinary skill in the art willrecognize that controller 28 can be used for other purposes. In anembodiment, controller 28 can use camera imaging device 20 to perform acolormetric analysis, which analyzes the concentration of a chemicalelement or chemical compound in a solution with the aid of a colorreagent. Controller 28 can quantify the amount of protein present in thefluid sample by measuring the absorption spectra and comparing it withprotein solutions of known concentration. In an embodiment, controller28 can analyze and interpret the results of colormetric protein assays,for example, a Bradford protein assay, a bichinchoninin acid assay (BCAassay), and/or a Ponceau S dye assay.

FIG. 8 illustrates an example embodiment of a control method that can beused by controller 28 to perform and analyze a reaction as describedherein, and FIG. 9 illustrates an example embodiment of a controller 28that can perform the method of FIG. 8. As illustrated, controller 28 caninclude a processor 250 and a memory 252, which can include anon-transitory computer readable medium. Memory 252 can include, forexample, an input module 254, a control module 256, an analysis module258, and an output module 260. Processor 250 can run the modules 252,254, 256, 258 in accordance with instructions stored on memory 252. Thebroken lines in FIG. 8 illustrate the electrical connections between themodules 252, 254, 256, 258 of controller 28 and various elements ofdevice 10. It should be understood by those of ordinary skill in the artthat the illustrated modules and/or additional modules can be connectedto the elements shown and/or additional elements.

The process begins by loading a test card 100 and/or a fluid sample intodevice 10. The fluid sample can be mixed with a reagent before injectioninto test card 100 and/or device 10, or can be mixed with a reagentwithin mixing chamber 126 of test card 100. In an embodiment, thereagent includes a PCR inhibitor-resistant polymerase along with aspecific mixture of reverse transcriptase (in the case of RNA targets)and surfactants/dispersants to allow for rapid sample dispersion andlysing. In an embodiment, the reagent mix can include, for example,olignucleotide primers, dNTP's DNA polymerase and other chemicals toassist the PCR. It is important to have a correct ratio of fluid sampleto final PCR volume, because if the correct ratio is not maintained, thePCR will take too long or fail.

Using user interface 60, a user can then choose a positive/negative testto run on the fluid sample. In an embodiment, the user can cycle througha plurality of tests on display 62 using buttons 64 and choose one ormore test to run. The plurality of tests can include, for example, a PCRanalysis, a cytometry analysis and/or an enzyme-linked immunosorbentassay (ELISA) analysis. In an alternative embodiment, a plurality ofdifferent types of test cards 100 can be inserted into device 10, witheach test card 100 corresponding to one or more specific test to be runon a fluid sample, and controller 28 can determine which test(s) to runby detecting the type of test card 100 inserted into device 10 withoutfurther instruction by the user. In another alternative embodiment,fluid microchannel 134 can be incorporated into device 10 rather thantest card 100, and a user can choose a test to run on the fluid sampleafter the fluid sample is injected directly into device 10.

Input module 254 is configured to receive the user inputs inputted intouser interface 60 and communicate the user inputs to control module 256.Input module 254 can also receive additional information via userinterface 60 and/or by the preprogramming of controller 28, for example,(i) real-time PCR crossover threshold value information; (ii) maximumfluorescence information; and (iii) melting curve inflection temperatureinformation. Other control parameters for the PCR reaction can also begiven. This includes the PCR denaturing, elongation and annealingtemperatures and dwell times.

Once a test card 100 and/or fluid sample has been loaded into device 10,control module 256 of controller 28 begins the control method at step200 by causing fluid actuation source 26 to pull fluid throughmicrochannel 134. In the illustrated embodiment, fluid actuation sourceapplies a negative pneumatic force to outlet port 130 via pneumatic tube40 to pull fluid through microchannel 134. In an alternative embodiment,fluid actuation source can include one or more other type of pump influid communication with microchannel 134.

After fluid actuation source 26 has been activated, but before thereaction begins, control module 256 at step 202 can verify that fluid islocated within target zone 166 by monitoring the capacitance ofmicrochannel 134 at one or more locations upstream and/or downstream oftarget zone 166. If fluid is detected in microchannel 134 upstreamand/or downstream of target zone 166, control module 256 can verify thatfluid is located within target zone 166, activate light source 22 atstep 204, and begin the reaction at step 206. Controller 28 can choose aportion of microchannel 134 to designate as target zone 166, or targetzone 166 can be predetermined.

In the illustrated embodiment, control module 256 begins the reaction atstep 206 by instructing power source 30 to send a current to electrodeslocated adjacent to target zone 166 via electrical contact device 26 tocause the fluid within target zone 166 to be heated. As the fluid sampleis heated, the nucleic acid molecules multiply by diffusion as explainedabove.

At the same time that the fluid sample is being heated within targetzone 166 so that the nucleic acid molecules multiply by diffusion,control module 256 at step 208 can cause camera imaging device 20 torecord a plurality of images of the reaction within target zone 166. Theplurality of images can then be sent to analysis module 208 for analysisat step 210. In an embodiment, test card 100 includes a transparentmaterial that allows images to be taken of target zone 166 of fluidmicrochannel 134 even though a layer of polymer material is locatedbetween camera imaging device 20 and fluid microchannel 134.

At step 210, analysis module 258 analyzes the images taken by cameraimaging device 20 to determine whether the fluid sample tests positiveor negative for a bacteria or virus. The type of analysis performed byanalysis module at step 210 will depend on the type of test being run onthe fluid sample.

If the assay being run on the fluid sample is a PCR analysis, thenanalysis module 258 can analyze the images as discussed above bymeasuring fluorescence based on detected wanted and unwanted objects.

If the assay being run on the fluid sample is a cytometry analysis, thenanalysis module 258 can also analyze the images as discussed above bymeasuring fluorescence based on detected wanted and unwanted objects.The cytometry analysis can differ from the PCR analysis, for example,because the fluid in target zone 166 does not need to be heated tomultiply molecules by diffusion, so step 206 can be skipped. With acytometry analysis, analysis module 258 can analyze the fluid samplewithin target zone 166, for example, by analyzing cell size, cell count,cell morphology (shape and structure), cell cycle phase, DNA content,and the existence or absence of specific proteins on cell surfaces. Inan embodiment, controller 28 can use various different fluorophores forflow cytometry. In an embodiment, to detect specific proteins on cellsurfaces, specifically designed fluorophores which bind to thoseproteins are mixed into the sample to cause the proteins of interest tofluoresce.

If the assay being run on the fluid sample is an ELISA analysis, thenagain the fluid in target zone 166 does not need to be heated tomultiply molecules by diffusion, so step 206 can be skipped. With anELISA analysis, analysis module 258 can analyze the fluid sample withintarget zone 166, for example, by measuring the concentration of ananalyte in the fluid sample using a colormetric analysis. In anembodiment, controller 28 can measure the amount of incident lightscattered on the ELISA target zone to determine the concentration of ananalyte. The method of measurement is the same as the turbiditymeasurement.

In an embodiment, controller 28 can perform genotyping tests as anextension a PCR.

At step 212, analysis module 258 determines based on the analysiswhether the fluid sample has tested positive or negative for a bacteriaor virus. The results of the analysis are then displayed on userinterface 60 by output module 260. In an embodiment, a simple “POSITIVE”or “NEGATIVE” indication can be displayed on user interface 60 to informwhether the fluid sample has tested positive or negative for a bacteriaor virus. In another embodiment, user interface 60 can display theresults for more than one bacteria or virus, or can display specificssuch as cell size, cell count, cell morphology (shape and structure),cell cycle phase, DNA content, and the existence or absence of specificproteins on cell surfaces. In an embodiment, controller 28 can displayviral titer in the case of a PCR reaction, or can display proteinconcertation and DNA concentration.

In an embodiment, the result of the analysis can be saved in a memorymodule of memory 252, so that the results can be reviewed at a latertime. If the result is saved, the result should be encoded to protectthe anonymity of the patient. In another embodiment, one or more encodedresults can be wirelessly transmitted for review at a location remotefrom diagnostic system 1.

In an embodiment, device 10 can include a global positioning system(GPS) sensor, and can record the result of the test along with a GPSsensor reading at the time of the test. Controller 28 can then aggregatea plurality of tests to determine viruses or bacteria that are moreprevalent in one area as opposed to another. In this embodiment,controller 28 does not need to save any patient information, and onlyneeds the GPS location and the number of positive and negative testresults at the location to determine the prevalence of the virus orbacteria at the location. The results can be used by a healthorganization to treat an area with appropriate medication for aprevalent virus or bacteria.

In another embodiment, the user can program the location into device 10before, during or after running a plurality of tests, and controller 28can aggregate the plurality of tests to determine viruses or bacteriathat are more or less prevalent in the programmed area.

In an embodiment, controller 28 can determine whether one or more ofyersina pestis, brucella, alphavirus, dengue virus and/or variola virusis present in the fluid sample.

The diagnostic system 1 disclosed herein can detect, for example,lentiviruses (ssRNA) and adenoviruses (dsDNA) in whole blood, as well asother infectious agents. Exemplary infectious agents which can bedetected by the system 1 disclosed herein include, but are not limitedto, bacterial pathogens, viral pathogens, fungal pathogens, and/orparasitic pathogens.

Exemplary non-limiting bacterial pathogens include Bacillus (e.g.,Bacillus anthracis, Bacillus cereus), Bartonella (e.g., Bartonellahenselae, Bartonella quintana), Bordatella (e.g., Bordatella pertussis),Borrelia (e.g., Borrelia burgdorferi, Borrelia garinii, Borreliaafzelii, Borrelia recurrentis), Brucella (e.g., Brucella abortus,Brucella canis, Brucella melitensis, Brucella suis), Campylobacter(e.g., Campylobacter jejuni), Chlamydia (e.g., Chlamydia pneumoniae,Chlamydia trachomatis), Chlamydophila (e.g., Chlamydophila psittaci),Clostridium (e.g., Clostridium botulinum, Clostridium difficile,Clostridium perfringens, Clostridium tetani), Corynebacterium (e.g.,Corynebacterium diphtheriae), Enterococcus (e.g., Enterococcus faecalis,Enterococcus faecium), Escherichia (e.g, Escherichia coli), Francisella(e.g., Francisella tularensis), Haemophilus (e.g., Haemophilusinfluenzae), Helicobacter (e.g., Helicobacter pylori), Legionella (e.g.,Legionella pneumophila), Leptospira (e.g., Leptospira interrogans,Leptospira santarosai, Leptospira weilii, Leptospira noguchii), Listeria(e.g., Listeria monocytogenes), Mycobacterium (e.g., Mycobacteriumleprae, Mycobacterium tuberculosis, Mycobacterium ulcerans), Mycoplasma(e.g., Mycoplasma pneumoniae), Neisseria (e.g., Neisseria gonorrhoeae,Neisseria meningitidis), Pseudomonas (e.g., Pseudomonas aeruginosa),Rickettsia (e.g., Rickettsia rickettsii), Salmonella (e.g., Salmonellatyphi, Salmonella typhimurium), Shigella (e.g., Shigella sonnei),Staphylococcus (e.g., Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus saprophyticus), Streptococcus (e.g., Streptococcusagalactiae, Streptococcus pneumoniae, Streptococcus pyogenes), Treponema(e.g., Treponema pallidum), Ureaplasma (e.g., Ureaplasma urealyticum),Vibrio (e.g., Vibrio cholerae), and Yersinia (e.g., Yersinia pestis,Yersinia enterocolitica, Yersinia pseudotuberculosis).

Exemplary non-limiting viral pathogens include Adenoviridae (e.g.,adenovirus), Herpesviridae (e.g., herpes simplex virus type 1 and type2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, humanherpesvirus type 8), Papillomaviridae (e.g., human papillomavirus),Polyomaviridae (e.g., BK virus, JC virus), Poxviridae (e.g., smallpox),Hepadnaviridae (e.g., hepatitis B virus), Parvoviridae (e.g., humanbocavirus, parvovirus B19), Astroviridae (e.g., human astrovirus),Caliciviridae, (e.g., Norwalk virus), Picornaviridae (e.g.,Coxsackievirus, hepatitis A virus, poliovirus, rhinovirus);Coronaviridae (e.g., severe acute respiratory syndrome virus, MiddleEast respiratory syndrome virus), Flaviviridae (e.g., hepatitis C virus,yellow fever virus, dengue virus, West Nile virus), Togaviridae (e.g.,rubella virus), Hepeviridae (e.g., hepatitis E virus), Retroviridae(e.g., lentiviruses, human immunodeficiency virus); Orthomyxoviridae(e.g., influenza virus), Arenaviridae (e.g., Guanarito virus, Juninvirus, Lassa virus, Machupo virus, Sabiá virus), Bunyaviridae (e.g.,Crimean-Congo hemorrhagic fever virus), Filoviridae (e.g., Ebola virus,Marburg virus), Paramyxoviridae (e.g., measles virus, mumps virus,parainfluenza virus, respiratory syncytial virus, human metapneumoniavirus, Hendra virus, Nipah virus), Phabdoviridae (e.g., rabies virus),Reoviridae (e.g., rotavirus, orbivirus, coltivirus, Banna virus), andunassigned viruses (e.g., Hepatitis D virus).

Exemplary non-limiting fungal pathogens include Candida (e.g., Candidaalbicans), Aspergillus (e.g., Aspergillus fumigatus, Aspergillusflavus), Crytopcoccus (e.g., Cryptococcus neoformans, Cryptococcuslaurentii, Cryptococcus gattii), Histoplasma (e.g., Histoplasmacapsulatum), Pneumocystis (e.g., Pneumocystis jirovecii, Pneumocystiscarinii), Stachybotrys (e.g., Stachybotrys chartarum).

Exemplary non-limiting parasitic pathogens include acanthamoeba,anisakis, Ascaris lumbricoides, botfly, Balantidium coli, bedbugs,Cestoda (tapeworm), chiggers, Cochliomyia hominivorax, Entamoebahistolytica, Fasciola hepatica, Giardia lamblia, hookworms, Leishmania,Linguatula serrata, liver flukes, Loa loa, Paragonimus—lung fluke,pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis,mites, tapeworms, Toxoplasma gondii, Trypanosoma, whipworms, andWuchereria bancrofti.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present subjectmatter and without diminishing its intended advantages. It is thereforeintended that such changes and modifications be covered by the appendedclaims.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” Accordingly,unless indicated to the contrary, the numerical parameters set forth inthe following specification and attached claims are approximations thatmay vary depending upon the desired properties sought to be obtained bythe present disclosure. At the very least, and not as an attempt tolimit the application of the doctrine of equivalents to the scope of theclaims, each numerical parameter should at least be construed in lightof the number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of the disclosure areapproximations, the numerical values set forth in the specific examplesare reported as precisely as possible. Any numerical value, however,inherently contains certain errors necessarily resulting from thestandard deviation found in their respective testing measurements.

The terms “a” and “an” and “the” and similar referents used in thecontext of the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g. “such as”) provided herein isintended merely to better illuminate the disclosure and does not pose alimitation on the scope of the disclosure otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the disclosure.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Groupings of alternative elements or embodiments of the disclosuredisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is hereindeemed to contain the group as modified thus fulfilling the writtendescription of all Markush groups used in the appended claims.

Preferred embodiments of the disclosure are described herein, includingthe best mode known to the inventors for carrying out the disclosure. Ofcourse, variations on those preferred embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects those of ordinary skill in the art toemploy such variations as appropriate, and the inventors intend for thedisclosure to be practiced otherwise than specifically described herein.Accordingly, this disclosure includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by thedisclosure unless otherwise indicated herein or otherwise clearlycontradicted by context.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the disclosure so claimed areinherently or expressly described and enabled herein.

Further, it is to be understood that the embodiments of the disclosuredisclosed herein are illustrative of the principles of the presentdisclosure. Other modifications that may be employed are within thescope of the disclosure. Thus, by way of example, but not of limitation,alternative configurations of the present disclosure may be utilized inaccordance with the teachings herein. Accordingly, the presentdisclosure is not limited to that precisely as shown and described.

The invention is claimed as follows:
 1. A device for analysing apolymerase chain reaction in a fluid sample, the device comprising: acurrent source configured to cause the polymerase chain reaction byheating the fluid sample within a target zone; a camera imaging deviceconfigured to record a plurality of images of the fluid sample in thetarget zone while the current source causes the polymerase chainreaction; and a controller configured to (i) distinguish wanted objectsin the plurality of images from an unwanted object in the plurality ofimages, and (ii) determine whether the fluid sample tests positive ornegative for a bacteria or virus based on the wanted objects.
 2. Thedevice of claim 1, wherein the wanted objects are nucleic acid moleculesand the unwanted object is an air bubble.
 3. The device of claim 1,wherein the controller is configured to analyze the plurality of imagesby dividing the target zone into a plurality of bins.
 4. The device ofclaim 3, wherein the plurality of bins are arranged in a grid with aplurality of rows and columns.
 5. The device of claim 3, wherein thecontroller is configured to determine whether the fluid sample hastested positive or negative for the bacteria or virus by selecting atleast two of the plurality of bins that overlap a cluster of wantedobjects and calculating a mean fluorescence value of the at least two ofthe plurality of bins.
 6. The device of claim 5, wherein the controlleris configured to exclude at least one of the plurality of bins from themean fluorescence value calculation if the at least one of the pluralityof bins overlaps the unwanted object.
 7. The device of claim 5, whereinthe controller is configured to assign a weight to at least one of theplurality of bins used in the mean fluorescence value calculation basedon the proximity of the at least one of the plurality of bins to theunwanted object.
 8. The device of claim 5, wherein the controller isconfigured to exclude at least one of the plurality of bins if the atleast one of the plurality of bins does not meet a minimum thresholdvalue for brightness.
 9. The device of claim 1, wherein the controlleris configured to report an inconclusive test if the controlleridentifies an unwanted image in the plurality of images.
 10. The deviceof claim 1, which includes a user interface, wherein the controllerincludes a plurality of preprogrammed analyses that can be performed onthe fluid sample, and wherein the user interface is configured to allowa user to select at least one analysis from the plurality ofpreprogrammed analyses.
 11. The device of claim 1, wherein the pluralityof images includes at least one of: (i) a plurality of still images ofthe polymerase chain reaction recorded by the camera imaging device overa period of time; or (ii) a video image of the polymerase chain reactionrecorded by the camera imaging device over the period of time.
 12. Thedevice of claim 1, wherein the controller is configured to distinguishthe wanted objects in the plurality of images from the unwanted objectin the plurality of images by comparing a size or shape of objects inthe plurality of images to an average size or shape of blood cells. 13.A device for analysing a polymerase chain reaction in a fluid sample,the device comprising: a current source configured to cause thepolymerase chain reaction by heating the fluid sample within a targetzone; a camera imaging device configured to record a plurality of imagesof the fluid sample in the target zone while the current source causesthe polymerase chain reaction; and a controller configured to analyzethe plurality of images by (i) dividing the target zone shown in theplurality of images into a plurality of bins, (ii) selecting at leasttwo of the plurality of bins, and (iii) calculating a fluorescence valueof the selected at least two of the plurality of bins.
 14. The device ofclaim 13, wherein the controller is configured to select the at leasttwo of the plurality of bins based on a threshold value for brightness.15. The device of claim 13, wherein the controller is configured toexclude at least one of the plurality of bins from the fluorescencevalue calculation if the at least one of the plurality of bins overlapsan unwanted object.
 16. The device of claim 13, wherein the controlleris configured to assign weights to the selected at least two of theplurality of bins for the calculation based on the proximity of theselected at least two of the plurality of bins to an unwanted object.17. A method of analysing a polymerase chain reaction in a fluid samplecomprising: heating the fluid sample in a target zone to cause thepolymerase chain reaction; recording a plurality of images of the fluidsample in the target zone during the polymerase chain reaction; dividingthe target zone into a plurality of bins; calculating a fluorescencevalue of at least two of the plurality of bins; and determining whetherthe fluid sample tests positive or negative for a bacteria or virusbased on the calculated fluorescence value.
 18. The method of claim 17,wherein calculating the fluorescence value includes weighting the atleast two of the plurality of bins based on proximity to an unwantedobject.
 19. The method of claim 17, wherein calculating the fluorescencevalue includes excluding at least one of the plurality of bins from thefluorescence value calculation if the at least one of the plurality ofbins overlaps an unwanted object.
 20. The method of claim 17, whereincalculating the fluorescence value includes excluding at least one ofthe plurality of bins based on a threshold value for brightness.
 21. Adevice for analysing a fluid sample, the device comprising: a cameraimaging device configured to record a plurality of images of the fluidsample while located within a target zone of a fluid microchannel; and acontroller configured to (i) distinguish wanted objects in the pluralityof images from an unwanted object in the plurality of images, and (ii)determine whether the fluid sample tests positive or negative for abacteria or virus based on the wanted objects.
 22. The device of claim21, wherein the controller is configured to determine whether the fluidsample tests positive or negative for the bacteria or virus by dividingthe target zone shown in the plurality of images into a plurality ofbins, selecting at least two of the plurality of bins, and calculating afluorescence value of the selected at least two of the plurality ofbins.